E. coli/gmeh3, an E. coli transformant expressing GmEH3, had the best regiocomplementarity (α_S = 72.4% and β_R = 97.6%) for (S)- and (R)-1,2-epoxyhexanes among five tested aliphatic chain rac-1,2-epoxides (1a–5a). To prepare (R)-hexane-1,2-diol (1b) with high ee_p via enantioconvergent hydrolysis of rac-1a, the regioselectivity coefficient α_S of GmEH3 for (S)-1a was improved by reshaping its substrate-binding pocket (SBP). Based on the semi-rational design, Trp¹⁰², Ile¹⁰⁵, Ile¹⁷⁸, Pro¹⁸⁷ and Leu¹⁸⁹ lining GmEH3’s SBP were identified, each of which was substituted with four residues, respectively. From 17 transformants harboring single-site variants of gmeh3, E. coli/gmeh3^W102L, /gmeh3^W102I, /gmeh3^W102V and /gmeh3^P187F were selected, catalyzing the conversion of rac-1a into (R)-1b with obviously enhanced ee_p values of 59.3–78.3%. Then, three transformants containing double-site variants were constructed by combinatorial mutagenesis of gmeh3^P187F separately with gmeh3^W102L, gmeh3^W102I and gmeh3^W102V. Among the three transformants, E. coli/gmeh3^W102V/P187F displayed the largest α_S of 89.7% with β_R of 96.2%. The enantioconvergent hydrolysis of 500 mM rac-1a was conducted using 200 mg/mL wet cells of E. coli/gmeh3^W102V/P187F at 25°C for 12 h, affording (R)-1b with 83.1% ee_p and 91.5% yield. The molecular docking simulation analysis demonstrated that GmEH3^W102V/P187F more regiopreferentially attacks C_α in the oxirane ring of (S)-1a than GmEH3.

E.coli / gmeh3是一种表达Gm EH3 的大肠杆菌转化体，在五个测试中 对 ( S )- 和 ( R )-1,2-环氧己烷具有最佳区域互补性（α _S  = 72.4% 和β _R = 97.6%）脂肪链外消旋-1,2-环氧化物 ( 1a – 5a )。为了通过rac - 1a的_对映聚合水解制备具有高ee _p的( R )-己烷-1,2-二醇( 1b ) ，Gm EH3的区域选择性系数α _S对于( S)- 1a通过重塑其底物结合袋 (SBP) 得到改进。基于半理性设计，色氨酸¹⁰²，异亮氨酸¹⁰⁵，异亮氨酸¹⁷⁸，脯氨酸¹⁸⁷和Leu ¹⁸⁹衬里了Gm EH3的SBP进行了鉴定，其中的每一个与四个残基分别被取代，。从携带gmeh3单位点变体的 17 个转化体中选择大肠杆菌/ gmeh3 ^W102L、/ gmeh3 ^W102I、/ gmeh3 ^W102V和/ gmeh3 ^P187F，催化rac - 1a转化为（R )- 1b具有明显增强的ee _p值 59.3-78.3%。然后，通过gmeh3 ^P187F分别与gmeh3 ^W102L、gmeh3 ^W102I和gmeh3 ^W102V的组合诱变构建了三个含有双位点变体的转化体。在三个转化体中，大肠杆菌/ gmeh3 ^W102V/P187F显示出最大的 α _S为 89.7%，β _R为 96.2%。使用 200 mg/mL大肠杆菌湿细胞进行500 mM rac -1a 的对映聚合水解/ gmeh3 ^W102V/P187F在 25°C 下保持 12 小时，得到 ( R ) -1b，具有 83.1% ee _p和 91.5% 产率。分子对接模拟分析证实了Gm EH3 ^{W102V / P187F}更regiopreferentially攻击Ç _α在（的环氧乙烷环小号） - 1A比了Gm EH3。